Development of Giant Unilamellar Vesicles for the Study of Crowded Protein Environments
David D. Gooray, Sandeep Dhall, and Victor G. J. Rodgers
Bioengineering Department, University of California, Riverside
Giant unilamellar vesicles (GUVs) are supramolecular structures consisting of amphiphiles that range in sizes from 10-100 µm.[1] They provide an easy method to: (1) observe environment for in-vitro studies of compartmentalized reactions and (2) model particular cell behavior. By studying the effects of crowded protein environments within GUVs, we will achieve a greater understanding of processes and properties such as mitochondrial swelling and osmotic pressure.
Phosphatidylcholine lipids are the only lipids that can form GUVs under electroformation.[2] GUVs with a diameter ranging between 50-100 µm will withstand micromanipulation techniques and allow us to study the effects of crowded protein environments.
We prepared GUVs using the 3 types of phosphatidylcholine lipids; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), L-α-phosphatidylcholine (Egg PC) and 1-2-dioleoyl-sn-glyeo-3-phosphocholine(DPC).
o A square chamber was cut from teflon and molded to fit the microscope slide.o Two cylindrical platinum wires (Sigma-Aldrich) of 1.0 mm diameter were housed
approximately parallel to each other with a 3.0 mm separationo Each wire was 1.5 mm from transparent viewing slideo All drilled holes and platinum-teflon interfaces were sealed with silicone
o Preparation of GUVs experimentso Obtain GUVs with 50-100 µm diametero Develop a crowded protein environment
Research Objectives
Conclusion & Future Work
Results (Continued)
Results
GUVs that were obtained were found attached to the electrode. They will be micromanipulated for studying crowded protein environments.
50 microns
25 microns
25 microns
POPC GUVs
25 microns
Egg PC GUVs
25 microns
DPC GUVs
Acknowledgements
oB2K Group, oDr. Vullev, Dr. Anvari, and Dr. ParkoJun Wang and Bioengineering department.
Electroformation Chamber
Function Generator
Inverted Microscope
Digital Imaging Camera
Oscilloscope
Pt Terminals
Silicone Sealant
Chamber designed by B2K
Teflon Housing
The GUVs selected for micromanipulation had an average size of 66.9 µm and were observed using a light microscope with a phase contrast filter. Average diameter of GUVs were determined using ImageJ. The average diameters of the GUVs obtained are:o POPC GUVs - 52.5 µm,o EPC GUVs - 67.6 µm, and o DOPC GUVs - 70.4 µm
We successfully setup the experiments for preparing GUVs and obtained the required sizes. GUVs with individual distribution on the wire were dependent on the precision of lipid application on the wire.
The future direction includes loading of the GUVs with proteins while surrounding their environments with a similar concentration of the protein. This is to reduce the osmotic pressure within the GUVs and their respective loading characteristics.
We will investigate better microinjection apparatus to enhance the precision of our piercings and load delivery into the GUVs.
Materials & MethodsYa
o Stock solutions of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), L-α-lysophosphatidylcholine (EPC) and 1-2-dioleoyl-sn-glyeo-3-phosphocholine (DPC) were made. (Avanti Polar Lipids)
o Frequency: 10 Hzo A-C Voltage Ramp: 0.3 V increased to 2.3 V
in 15 mino 2.3 V applied for 105 min
[1]Luisi, P. and Walde, P. Giant Vesicles. New York: John Wiley & Sons Ltd. 2000
[2]Angelova,M.I., S.Soleau, P.Melead,J.- Faucon, and P.Bothorel. Preperation of giant vesicles by external ac electric fields: kinetics and applications.Prog. Colloid Polym. Sci. 89:127-131(1992)
Experimental Setup
References
Single GUV
Experimental set up Size distribution of GUVs
Introduction
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